PROJECT SUMMARY STING-mediated type I interferon (IFN) signaling plays an important role in innate immune response to infections. The STING pathway is unique in that it requires ER-to-ERGIC/Golgi translocation to activate downstream signaling. Gain-of-function mutations in TMEM173 encoding STING have been reported in STING- associated vasculopathy with onset in infancy (SAVI). We showed that these mutants constitutively activate STING trafficking and signaling independent of ligand binding. In addition to IFN, STING also drives other cellular and physiological responses that do not appear to require IFN signaling, such as T cell proliferation and survival. How are these new STING functions engaged remains unknown. Limited evidence thus far suggests that NFkB and STING trafficking are involved. Here, we propose to study STING activation and signaling that regulate the survival of T cells. The proposed study is based on our recent findings from characterizing the Sting-N153S (N154S in human STING) knock-in mouse, which develop many pathological features of the human disease, including inflammation within the lung and T cell cytopenia. N153S mice develop disease independent of IRF3/IFN, raising the possibility that non-IFN functions of STING is responsible for disease pathology in the N153S mouse. In our preliminary studies, we mapped STING-mediated IFN and NFkB signaling motifs to distinct regions of the C-terminus. Inducible expression of N154S in human Jurkat T cells also caused cell death. Thus, we hypothesize that disease-associated STING mutations such as N154S constitutively translocates from ER- to-ERGIC/Golgi and chronically activates a novel NFkB or unknown signaling cascade leading to T cell death. We will first determine how STING signaling is activated by ER-to-ERGIC/Golgi translocation, a mechanism shared by both ligand-dependent and ?independent activation of STING. Then, we will investigate how STING activation in T cells induces cell death, using engineered human T cells and the gain-of-function Sting-N153S mouse. Lastly, we will study a new knock-in mouse Sting-S365A, which disrupts STING's ability to activate IRF3/IFN while retaining other signaling capabilities, under a variety of physiological conditions such as infection, cell proliferation and cell death. Studies proposed here will establish a new paradigm of STING biology by characterizing an important new function of STING that regulates T cell survival under physiologically relevant disease settings.